CN115491322B - Extraction method and application of alkaloid - Google Patents

Extraction method and application of alkaloid Download PDF

Info

Publication number
CN115491322B
CN115491322B CN202210562230.1A CN202210562230A CN115491322B CN 115491322 B CN115491322 B CN 115491322B CN 202210562230 A CN202210562230 A CN 202210562230A CN 115491322 B CN115491322 B CN 115491322B
Authority
CN
China
Prior art keywords
compound
compounds
methanol
component
streptomyces
Prior art date
Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
Active
Application number
CN202210562230.1A
Other languages
Chinese (zh)
Other versions
CN115491322A (en
Inventor
张应烙
宋涛
吴俊�
项子琳
Current Assignee (The listed assignees may be inaccurate. Google has not performed a legal analysis and makes no representation or warranty as to the accuracy of the list.)
Anhui Agricultural University AHAU
Original Assignee
Anhui Agricultural University AHAU
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Anhui Agricultural University AHAU filed Critical Anhui Agricultural University AHAU
Priority to CN202210562230.1A priority Critical patent/CN115491322B/en
Publication of CN115491322A publication Critical patent/CN115491322A/en
Application granted granted Critical
Publication of CN115491322B publication Critical patent/CN115491322B/en
Active legal-status Critical Current
Anticipated expiration legal-status Critical

Links

Classifications

    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
    • C12N1/20—Bacteria; Culture media therefor
    • C12N1/205—Bacterial isolates
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N37/00—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids
    • A01N37/18—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof
    • A01N37/22—Biocides, pest repellants or attractants, or plant growth regulators containing organic compounds containing a carbon atom having three bonds to hetero atoms with at the most two bonds to halogen, e.g. carboxylic acids containing the group —CO—N<, e.g. carboxylic acid amides or imides; Thio analogues thereof the nitrogen atom being directly attached to an aromatic ring system, e.g. anilides
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N43/00—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds
    • A01N43/72—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms
    • A01N43/84—Biocides, pest repellants or attractants, or plant growth regulators containing heterocyclic compounds having rings with nitrogen atoms and oxygen or sulfur atoms as ring hetero atoms six-membered rings with one nitrogen atom and either one oxygen atom or one sulfur atom in positions 1,4
    • A—HUMAN NECESSITIES
    • A01—AGRICULTURE; FORESTRY; ANIMAL HUSBANDRY; HUNTING; TRAPPING; FISHING
    • A01N—PRESERVATION OF BODIES OF HUMANS OR ANIMALS OR PLANTS OR PARTS THEREOF; BIOCIDES, e.g. AS DISINFECTANTS, AS PESTICIDES OR AS HERBICIDES; PEST REPELLANTS OR ATTRACTANTS; PLANT GROWTH REGULATORS
    • A01N55/00—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur
    • A01N55/02—Biocides, pest repellants or attractants, or plant growth regulators, containing organic compounds containing elements other than carbon, hydrogen, halogen, oxygen, nitrogen and sulfur containing metal atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/16—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms
    • C07C233/24—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C07C233/25—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by singly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring having the carbon atom of the carboxamide group bound to a hydrogen atom or to a carbon atom of an acyclic saturated carbon skeleton
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07C—ACYCLIC OR CARBOCYCLIC COMPOUNDS
    • C07C233/00—Carboxylic acid amides
    • C07C233/01—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms
    • C07C233/30—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms
    • C07C233/33—Carboxylic acid amides having carbon atoms of carboxamide groups bound to hydrogen atoms or to acyclic carbon atoms having the nitrogen atom of at least one of the carboxamide groups bound to a carbon atom of a hydrocarbon radical substituted by doubly-bound oxygen atoms with the substituted hydrocarbon radical bound to the nitrogen atom of the carboxamide group by a carbon atom of a six-membered aromatic ring
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07D—HETEROCYCLIC COMPOUNDS
    • C07D265/00—Heterocyclic compounds containing six-membered rings having one nitrogen atom and one oxygen atom as the only ring hetero atoms
    • C07D265/28—1,4-Oxazines; Hydrogenated 1,4-oxazines
    • C07D265/34—1,4-Oxazines; Hydrogenated 1,4-oxazines condensed with carbocyclic rings
    • C07D265/38—[b, e]-condensed with two six-membered rings
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07F—ACYCLIC, CARBOCYCLIC OR HETEROCYCLIC COMPOUNDS CONTAINING ELEMENTS OTHER THAN CARBON, HYDROGEN, HALOGEN, OXYGEN, NITROGEN, SULFUR, SELENIUM OR TELLURIUM
    • C07F15/00—Compounds containing elements of Groups 8, 9, 10 or 18 of the Periodic System
    • C07F15/02—Iron compounds
    • C07F15/025—Iron compounds without a metal-carbon linkage
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/008—Preparation of nitrogen-containing organic compounds containing a N-O bond, e.g. nitro (-NO2), nitroso (-NO)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P13/00—Preparation of nitrogen-containing organic compounds
    • C12P13/02—Amides, e.g. chloramphenicol or polyamides; Imides or polyimides; Urethanes, i.e. compounds comprising N-C=O structural element or polyurethanes
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12P—FERMENTATION OR ENZYME-USING PROCESSES TO SYNTHESISE A DESIRED CHEMICAL COMPOUND OR COMPOSITION OR TO SEPARATE OPTICAL ISOMERS FROM A RACEMIC MIXTURE
    • C12P17/00—Preparation of heterocyclic carbon compounds with only O, N, S, Se or Te as ring hetero atoms
    • C12P17/14—Nitrogen or oxygen as hetero atom and at least one other diverse hetero ring atom in the same ring
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12R—INDEXING SCHEME ASSOCIATED WITH SUBCLASSES C12C - C12Q, RELATING TO MICROORGANISMS
    • C12R2001/00—Microorganisms ; Processes using microorganisms
    • C12R2001/01—Bacteria or Actinomycetales ; using bacteria or Actinomycetales
    • C12R2001/465—Streptomyces

Abstract

The invention relates to the technical field of microbial engineering, in particular to an alkaloid compound extracted from a liquid fermentation product of streptomyces nigromaculatus body surface (Streptomyces tanashiensis) BYF112, and a preparation method and application thereof. Specifically, the invention discloses 11 alkaloid compounds, which have the structural formula:wherein is converted intoCompounds 1,2,3 and 7 are novel compounds. The alkaloid compound can be prepared from streptomyces (Streptomyces tanashiensis) BYF112 with a preservation number of CCTCC M2019474. The alkaloid compounds 1,2, 5, 6, 10 and 11 can be used as anticancer drugs, specifically, the compounds 2, 5, 6, 10 and 11 can inhibit the growth of human malignant melanoma cells (A375), the compounds 1, 5, 6 and 11 can inhibit the growth of ovarian cancer cells (SKOV-3), and the compounds 5, 6, 10 and 11 can inhibit the growth of human gastric cancer cells (MGC-803); compound 11 is useful as an antibacterial agent, in particular, inhibiting the growth of streptococcus equi; compounds 5 and 11 are useful as herbicides, in particular, inhibiting barnyard grass root growth.

Description

Extraction method and application of alkaloid
Technical Field
The invention relates to the technical field of microorganisms, in particular to a preparation method and application for extracting alkaloid from Streptomyces nigromaculatus body surface (Streptomyces tanashiensis) BYF112 liquid fermentation product.
Background
At present, most of the chemotherapeutic drugs used clinically have large toxic and side effects, and tumor cells are influenced by the drug resistance of the tumor cells and external drugs, so that a large number of tumor multi-drug resistant cell strains appear, and the curative effect of the chemotherapeutic drugs is greatly reduced; on the other hand, due to the increasingly serious phenomenon of abuse of antibiotics, the effect of antibiotics which are originally specific in clinic on drug-resistant bacteria is smaller and smaller. If this phenomenon cannot be improved, it will pose a great threat to global health safety. Therefore, it is urgent to find a novel lead compound having anti-tumor or antibiotic activity.
Along with the deep understanding of toxic and side effects of synthetic chemicals and the advocating of natural attitudes in the global scope, the international importance of natural medicines is continuously enhanced, and the natural medicines have the characteristics of high efficiency and low toxicity, so the natural medicines are a great trend of future medicine development.
Streptomyces is the most bulky member of the order Actinomycetales, gram-positive, aerobic, filamentous bacteria, widely distributed in the natural environment, and produces secondary metabolites of various biological activities, such as antibiotics, vitamins, enzyme inhibitors, and the like. More than 60% of clinical antibiotics are statistically derived from streptomyces. In recent years, research has found that streptomycete in insect symbiotic bacteria special habitat has great potential of being used as medicinal resources, such as special metabolic products, novel chemical structure, strong biological activity and the like. For example, a novel bisindole anti-cancer agent Lei Beika mycin was isolated from a strain of Pseudomonas (Van Arnam, E.B.; ruzzini. A.C.; sit, C.S.; horn, H.; pinto-T, A.A.; currie, C.R.; clardy, J.J.Am. Chem. Soc.2015,137, 14272-14274).
Disclosure of Invention
The invention aims to solve the technical problem of providing a Streptomyces nigromaculatus symbiotic (Streptomyces tanashiensis) BYF112 and a preparation and application of an anticancer active compound generated by the same, which are taken as an anticancer lead compound.
In order to solve the technical problems, the invention provides an anticancer active compound generated by streptomyces, which has the structural formula as follows:
the invention also provides streptomycete BYF-112 with strong anticancer activity, the preservation number is CCTCC M2019474, the preservation date is 2019, 06 and 20 days, and the preservation address is eight paths of Lopa nationality in Wuchang district of Wuhan, hubei province of China Center for Type Culture Collection (CCTCC).
The invention also provides a preparation method of the novel phenazine anticancer active metabolite, which comprises the following steps:
1) Streptomyces BYF-112 with the preservation number of CCTCC M2019474 is inoculated on a Gao's medium and cultured on a shaking table for 2-3 days under the conditions of 160-200rpm (preferably 180 rpm) and 27.5-28.5 ℃ (preferably 28 ℃) to obtain seed liquid;
2) Inoculating the seed solution into a Gao's culture medium, and fermenting for 6.5-7.5 days (preferably 7 days) under the conditions of 160-200rpm (preferably 180 rpm) and 27.5-28.5 ℃ (preferably 28 ℃);
generally, each 10mL of seed solution is inoculated into 350-450 mL (preferably 400mL of Gao's medium);
3) Filtering the fermentation liquor obtained in the step 2) (filtering by two layers of gauze), extracting the filtrate by ethyl acetate (total extraction is carried out for 3 times, the dosage of ethyl acetate each time=the volume of the fermentation liquor), concentrating and drying in vacuum (the vacuum degree of 0.1 negative pressure and the drying at 45 ℃ for 30-50 minutes) to obtain extractum (black);
4) Subjecting the extract obtained in the step 3) to silica gel column chromatography segmentation, and performing gradient elution by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 100:2, 100:4 and 100:8; thereby obtaining 5 elution fractions F1 to F5, respectively;
5) Component F2 obtained in step 4) was dried, recrystallized from methanol and analyzed by TLC to give compound 8.
6) Drying the component F3 obtained in the step 4), recrystallizing in methanol, performing TLC analysis to obtain a compound 11 and a component F3-1, drying the component F3-1, recrystallizing in ethyl acetate, performing TLC analysis to obtain a compound 5 and a compound 10 and a component F3-1-1 respectively, separating the component F3-1-1 by gel column chromatography, and performing TLC analysis to obtain a compound 2 by using methanol as an eluent.
7) Drying the component F4 obtained in the step 4), recrystallizing in methanol, analyzing by TLC to obtain a compound 9 and components F4-1, F4-2, F4-3 and F4-4, combining the components F4-1 and F4-2, separating by gel column chromatography, using methanol as an eluent, and analyzing by TLC to obtain compounds 1,6 and 7 respectively; gradient elution is carried out on the component F4-3 by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 50:1, 20:1 and 10:1; drying dichloromethane and methanol in a volume ratio of 10:1, recrystallizing in methanol, and performing TLC analysis to obtain a compound 3; and (3) carrying out gradient elution on the component F4-4 by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 100:2, 100:4 and 100:8, drying the component F4-4 by adopting the volume ratio of the dichloromethane to the methanol of 100:4, and recrystallizing the component F4-4 in the methanol, and carrying out TLC analysis to obtain the compound 4.
Further, in the step (1), the streptomyces (Streptomyces tanashiensis) BYF-112 is activated by a Gao medium and inoculated in a Gao liquid medium for fermentation culture for 6.5-7.5 d at a temperature of 27.5-28.5 ℃ (preferably 28 ℃), and a rotation speed of 160-200rpm (preferably 180 rpm).
The Gao's liquid culture medium used in the invention is 20g of soluble starch and 0.5g of KNO 3 ,0.5g K 2 HPO 4 ·3H 2 O,0.5g MgSO 4 ·7H 2 O,0.5g NaCl,0.01g FeSO 4 ·7H 2 O g, 1L of distilled water is used for preparing the pH value of 7.0-8.0.
The invention also provides an application of the secondary metabolite in anticancer, and the compound can be used as a prepared anti-tumor, antibacterial and herbicidal medicament.
The compound can be used as a medicament for inhibiting the growth of melanoma, gastric cancer, breast cancer and ovarian cancer cells, inhibiting the growth of streptococcus equi and inhibiting the growth of barnyard grass roots.
The Streptomyces of the invention is named Streptomyces tanashiensis BYF-112 in taxonomy, and is separated from suburban areas of Jiangsu river in China, and the strain is preserved in China Center for Type Culture Collection (CCTCC) M2019474 in the 6 th month and 20 th day of 2019. The preservation unit address is China, wuhan.
The invention has the following beneficial effects:
1. the secondary metabolite (1-4 and 7) of the streptomyces BYF-112 is a novel compound, and (5-6, 8-11) is a known compound, and can be used as a lead compound for inhibiting the cell growth of melanoma, gastric cancer, breast cancer and ovarian cancer, inhibiting the growth of streptococcus tetrandra and inhibiting the growth of barnyard grass roots.
2. The streptomyces BYF-112 secondary metabolite can be produced by liquid fermentation by utilizing microorganisms, and has the advantages of simple process, short period, low cost and guaranteed source.
3. The invention synthesizes the secondary metabolite of streptomycete BYF-112 by using a biological method, and has no pollution to the environment.
Preservation information
Preservation time: 2019, 6 and 20 days
Preservation unit: china center for type culture Collection;
preservation number: cctccc M2019474;
deposit unit address: eight paths of universities of Lopa nationality at mountain and Wuhan in Wuhan, hubei province of China Center for Type Culture Collection (CCTCC);
classification naming: streptomyces tanashiensis BYF-112.
Drawings
FIG. 1 shows the structural formulas of 11 alkaloid compounds provided by the embodiment of the invention.
FIG. 2 shows the compound vegfrecine A (1) according to the embodiment of the invention 1 H NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 3 shows the compound vegfrecine A (1) according to the embodiment of the invention 13 C NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 4 is a chart of COSY (Agilent DD2, DMSO-d 6.) spectrum of the compound vegfrecine A (1) provided in the example of the present invention.
FIG. 5 is a spectrum of HSQC (Agilent DD2, DMSO-d 6.) of the compound vegfrecine A (1) provided in the example of the present invention.
FIG. 6 is a DEPT (Agilent DD2, DMSO-d 6.) spectrum of the compound vegfrecine A (1) provided in the example of the present invention.
FIG. 7 is a spectrum of HMBC (Agilent DD2, DMSO-d 6.) of the compound vegfrecine A (1) provided in the example of the present invention.
FIG. 8 is a HR-ESI-MS diagram of the compound vegfrecine A (1) according to an example of the present invention.
FIG. 9 shows the compound vegfrecine B (2) according to the embodiment of the present invention 1 H NMR (Agilent DD2, actone-d 6.) spectra.
FIG. 10 shows the compound vegfrecine B (2) according to the embodiment of the present invention 13 C NMR (Agilent DD2, actone-d 6.) spectra.
FIG. 11 is a spectrum of COSY (Agilent DD2, actone-d 6.) of the compound vegfrecine B (2) provided in the example of the present invention.
FIG. 12 is a spectrum of HSQC (Agilent DD2, actone-d 6.) of the compound vegfrecine B (2) provided in the example of the present invention.
FIG. 13 is a spectrum of DEPT (Agilent DD2, actone-d 6.) of the compound vegfrectine B (2) provided in the example of the present invention.
FIG. 14 is a spectrum of HMBC (Agilent DD2, actone-d 6.) of the compound vegfrecine B (2) provided in the example of the present invention.
FIG. 15 is a HR-ESI-MS diagram of the compound vegfrecine B (2) according to an example of the present invention.
FIG. 16 shows an example of the present invention of exfolidazone A (3) 1 H NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 17 shows an example of the present invention of exfolidazone A (3) 13 C NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 18 is a chart of the COSY (Agilent DD2, DMSO-d 6.) spectrum of the compound exfoliazone A (3) provided in the examples of the present invention.
FIG. 19 is a spectrum of HSQC (Agilent DD2, DMSO-d 6.) of the compound exfoliazone A (3) provided in the examples of the present invention.
FIG. 20 is a spectrum of DEPT (Agilent DD2, DMSO-d 6.) of the compound exfoliazone A (3) provided in the examples of the present invention.
FIG. 21 is a chart of the HMBC (Agilent DD2, DMSO-d 6.) spectrum of the compound exfoliazone A (3) provided in the examples of the present invention.
FIG. 22 is a HR-ESI-MS of the compound exfolidazone A (3) provided in the examples of the present invention.
FIG. 23 shows a compound venezueline H (7) provided in an example of the present invention 1 H NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 24 is a diagram of venezueline H (7) as a compound according to an embodiment of the present invention 13 C NMR (Agilent DD2, DMSO-d 6.) spectra.
FIG. 25 is a chart of the COSY (Agilent DD2, DMSO-d 6.) spectrum of the compound venezueline H (7) provided in the examples of the present invention.
FIG. 26 is a spectrum of HSQC (Agilent DD2, DMSO-d 6.) of the compound venezueline H (7) provided in the examples of the present invention.
FIG. 27 is a spectrum of HMBC (Agilent DD2, DMSO-d 6.) of the compound venezueline H (7) provided in the examples of the present invention.
FIG. 28 is a spectrum of HMBC (Agilent DD2, DMSO-d 6.) of the compound venezueline H (7) provided in the examples of the present invention.
FIG. 29 is a HR-ESI-MS plot of the compound venezueline H (7) provided in the examples of the present invention.
FIG. 30 shows the results of a test for inhibition of barnyard grass root growth by the compounds of the present invention
Detailed Description
For a better understanding of the present invention, reference will be made to the following description of specific examples, but the protection of this patent is not limited thereto.
The invention will be further explained with reference to specific examples.
Example 1: separation of Streptomyces (Streptomyces tanashiensis) BYF-112 and separation of purified Streptomyces BYF-112:
the black wing soil termites were starved for 24h before separation, and young workers (20 heads) were removed with sterile forceps and placed in a sterile centrifuge tube. 1mL of sterile PBS buffer pH7.4 was added and the mixture was shaken to give a rinse solution. Diluting the shaking solution with sterile water to 10 -1 、10 -2 、10 -3 0.1mL of each gradient dilution was spread on a plate of M3 medium, and cultured in an incubator at 28℃for 4 days. After colony grows out, a small amount of hypha is picked from the edge of the colony of the tissue block, transferred to a Gao's medium plate again, purified to obtain single colony, and identified by morphology and molecular biology as Streptomyces fraxinus (Streptomyces tanashiensis) BYF-112
Culture medium: 20g of soluble starch, 0.5g of KNO 3 ,0.5g K 2 HPO 4 ·3H 2 O,0.5g MgSO 4 ·7H 2 O,0.5g NaCl,0.01g FeSO 4 ·7H 2 O, preparing 1L with distilled water, sterilizing at 121 ℃ for 20min under the pH of 7.0-8.0,1.1 atm (conventional sterilization).
The Streptomyces (Streptomyces tanashiensis) BYF-112 was deposited in the following depository: china center for type culture Collection; the preservation name is: streptomyces tanashiensis BYF-112, deposit address: chinese university of armed chinese; preservation date: 2019.06.20; the preservation number is CCTCC M2019474.
The streptomycete (Streptomyces tanashiensis) BYF-112 can be inoculated into a Golgi inclined surface test tube for storage.
Example 2: liquid fermentation of insect symbiotic bacteria-Streptomyces avium (Streptomyces tanashiensis) BYF-112
Fresh mycelium blocks (about 2-3 g) of Streptomyces (Streptomyces tanashiensis) BYF-112 are inoculated into 250mL conical flasks, each flask contains 150mL of Gao's medium, 10-15 flasks are inoculated onto a shaker and cultured for 2-3 days at 180rpm and 28 ℃ as seed solution, then 10mL of seed solution is inoculated into 1000mL conical flasks filled with 400mL of Gao's medium and fermented for 7 days at 180rpm and 28 ℃.
Remarks description: after the Streptomyces fraxinus (Streptomyces tanashiensis) BYF-112 is activated by a conventional Gao's medium, the fresh Streptomyces fraxinus (Streptomyces tanashiensis) BYF-112 can be obtained.
The formula of the Gao's culture medium is as follows: 20g of soluble starch, 0.5g of KNO 3 ,0.5g K 2 HPO 4 ·3H 2 O,0.5g MgSO 4 ·7H 2 O,0.5g NaCl,0.01g FeSO 4 ·7H 2 O, preparing 1L with distilled water, sterilizing at 121 ℃ for 20min under the pH of 7.0-8.0,1.1 atm (conventional sterilization).
EXAMPLE 3 extraction and isolation of Streptomyces BYF-112 secondary metabolite
The 15.6L fermentation broth prepared in example 2 was filtered through two layers of gauze, the filtrate was extracted 3 times with ethyl acetate (the amount of ethyl acetate was 50L each time), and the obtained extract was concentrated and dried in vacuo (dried at 45℃for 30-50 minutes at a vacuum of 0.1 negative pressure) to obtain a black extract. Subjecting the extract to silica gel column chromatography segmentation (the silica gel column adopts 200-300 meshes of silica gel, and the weight is 200 g), wherein the volume ratio of dichloromethane to methanol is 100:0, 100:1, 100:2, 100:4 and 100:8 in sequence; thereby obtaining 5 elution fractions F1 to F5, respectively; the dosage of each eluent is 2700-3000, 1200-1400, 5600-5900, 2000-2300, 1200-1500, 1800-2000 and 800-1200 ml respectively; thereby obtaining 5 elution fractions F1 to F5, respectively;
the obtained component F2 was dried, recrystallized from methanol and analyzed by TLC to obtain compound 8. The obtained component F3 is dried and recrystallized in methanol, the compound 11 and the component F3-1 are obtained through TLC analysis, the component F3-1 is dried and recrystallized in ethyl acetate, the compounds 5 and 10 and the component F3-1-1 are respectively obtained through TLC analysis, the component F3-1-1 is separated through gel column chromatography, and the compound 2 is obtained through TLC analysis by taking methanol as an eluent. Drying the obtained component F4, recrystallizing in methanol, analyzing by TLC to obtain a compound 9 and components F4-1, F4-2, F4-3 and F4-4, combining the components F4-1 and F4-2, separating by gel column chromatography, and analyzing by TLC with methanol as eluent to obtain compounds 1,6 and 7 respectively; gradient elution is carried out on the component F4-3 by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 50:1, 20:1 and 10:1; drying dichloromethane and methanol in a volume ratio of 10:1, recrystallizing in methanol, and performing TLC analysis to obtain a compound 3; and (3) carrying out gradient elution on the component F4-4 by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 100:2, 100:4 and 100:8, drying the component F4-4 by adopting the volume ratio of the dichloromethane to the methanol of 100:4, and recrystallizing the component F4-4 in the methanol, and carrying out TLC analysis to obtain the compound 4.
The structural formula is as follows:
EXAMPLE 4 structural resolution of Streptomyces avium BYF-112 metabolite
Compound 1 is a mauve powder, HR-ESI-MS at m/z 323.0642[ M+Na ]] + Molecular ion peaks are given (as shown in FIG. 8), and the molecular formula of the presumed compound is C 15 H 12 N 2 O 5 . For the compound 1 H-NMR (as shown in FIG. 2) and 13 C-NMR spectrum (shown in FIG. 3) analysis shows that the compound has the same parent nucleus structure as Vegfrectine, and the structure of the compound is finally determined as shown in formula (1) through two-dimensional HSQC (shown in FIG. 5), COSY (shown in FIG. 4) and HMBC (shown in FIG. 7) spectrum analysis. Compound 1 is designated VegfrecineA (1), a compound 1 H and 13 the assignment of C NMR (DMSO-d 6) is shown in Table 1.
Compound 2 is a mauve powder, HR-ESI-MS at m/z 325.0975[ M+Na ]] + Molecular ion peaks are given (as shown in FIG. 15), and the molecular formula of the presumed compound is C 15 H 14 N 2 O 5 . For the compound 1 H-NMR (as shown in FIG. 9) and 13 analysis of the C-NMR spectrum (shown in FIG. 10) revealed that the compound 1 had the same parent nucleus structure, the only difference being that the benzene ring 5 was oxidized to aldehyde group. The structure of the compound was finally determined as shown in formula (1) by spectrogram analysis of two-dimensional HSQC (shown in fig. 12), COSY (shown in fig. 11) and HMBC (shown in fig. 14). Compound 2 is designated VegfrecineB (2), a compound 1 H and 13 the assignment of C-NMR (Acetone-d 6) is shown in Table 1.
Compound 3 is a red solid, HR-ESI-MS at m/z 257.0930[ M+H ]] + Molecular ion peaks are given (as shown in FIG. 22), and the molecular formula of the presumed compound is C 14 H 12 N 2 O 3 . For the compound 1 H-NMR (as shown in FIG. 16) and 13 C-NMR spectrum (shown in FIG. 17) analysis shows that the compound has the same parent nucleus structure as exfolidazone, and the structure of the compound is finally determined as shown in formula (1) through two-dimensional HSQC (shown in FIG. 19), COSY (shown in FIG. 18) and HMBC (shown in FIG. 21) spectrum analysis. Compound 3 is designated as exfolidazone A (3), a compound 1 H and 13 the assignment of C NMR (DMSO-d 6) is shown in Table 2.
Compound 7 is a red powder, HR-ESI-MS at m/z 470.1320[ M+Na ]] + Molecular ion peaks are given (as shown in FIG. 29), and the molecular formula of the presumed compound is C 24 H 21 N 3 O 6 . For the compound 1 H-NMR (as shown in FIG. 23) and 13 C-NMR spectrum (shown in FIG. 24) analysis shows that the compound has the same parent nucleus structure as Venezueline, and the structure of the compound is finally determined as shown in formula (1) through two-dimensional HSQC (shown in FIG. 26), COSY (shown in FIG. 25) and HMBC (shown in FIG. 28) spectrum analysis. Compound 7 is designated VegfrecineA (7), a compound 1 H and 13 the assignment of C NMR (DMSO-d 6) is shown in Table 3.
TABLE 1 Compounds 1 (VegfrecineA, DMSO-d 6) and 2 (VegfrecineB, acetone-d 6) 1 H and 13 CNMR attribution
TABLE 2 Compound 3 (Exfolidazonea) 1 H and 13 CNMR (DMSO-d 6) is attributed.
TABLE 3 Compound 7 (Venezueline H) 1 H and 13 CNMR (DMSO-d 6) is attributed.
Determination of the cytotoxic Activity of Compounds of example 5
Culturing the cells in DMEM culture medium containing 10% fetal calf serum, culturing in a 5% carbon dioxide cell incubator at 37 deg.C, transferring stably for 2-3 generations, and taking cells in logarithmic growth phase for subsequent experiment.
Cell proliferation experiments the log phase cells were collected, counted, cell suspension concentration was adjusted, and the cells were seeded at 4000/100 μl/well in 96 well plates and incubated at 37deg.C under 5% carbon dioxide for 24h until cell monolayers were 2/3 of the well bottom area. Medium was carefully aspirated from the well edges, and each was incubated for 24, 48, 72h with a concentration gradient of compound, 100 μl/well, 4 parallel wells per group. Under the dark condition, adding 20 μl/well of MTT solution,the mixture is placed into an incubator for culturing for 4 hours. The in-well culture broth was carefully aspirated, DMSO was added, 150. Mu.l/well, and the mixture was placed on a shaker for 15min at low speed. Absorbance (OD) values of each well were measured at 492nm in a microplate reader to control Kong Diaoling. Results were recorded and cell inhibition was calculated as inhibition (Inhibitory concentration, IC) (%) = (1- (experimental OD mean-blank OD mean)/(control OD mean-blank OD mean)]100% the experiment was repeated 3 times and the mean and median Inhibitory Concentration (IC) 50 ). The positive control was doxorubicin (Adriamycin) and the blank control was solvent-treated tumor cells.
TABLE 4 IC of Streptomyces BYF-112 secondary metabolite 50
Doxorubicin: positive control. b, not tested. C, concentration >100 μm. A375: human malignant melanoma cells. SKOV-3, human ovarian cancer cells. MDA-MB231: human breast cancer cells. MGC-803: human gastric cancer cells. L-02: normal cells.
As shown in Table 4, pair SKOV-3IC in New Compound 1 50 The value is 76.18+/-2.09 mu M, and compared with the positive control of 0.72+/-0.12 mu M, the cytotoxic activity is extremely weak, but the toxic activity on normal cells is also very weak; the novel compound 2 has an IC50 value of 47.25+/-0.98 mu M for A375, and has weaker cytotoxic activity than the positive control of 1.01+/-0.48 mu M, but stronger cytotoxic activity for SKOV-3 than the compound 1 and weaker cytotoxic activity for normal cells; the novel compound 3 was less toxic to four cell lines. Compounds 5, 6, 11 showed significant activity on A375, IC 50 The values were 7.32, 5.85, 5.46 and 8.20 μm, respectively, which is comparable to the positive control ic50=1.01 μm, while compound 11 was not toxic to normal cells (L-02). In addition, compounds 5 and 6 show better toxicity activity on SKOV-3 and IC 50 The value is 6.09-11.09 mu M, and the positive control doxorubicin IC 50 The value of 0.72.+ -. 0.12. Mu.M cytotoxicity was comparable. Compound 6,8,9,10 has certain activity on A375 and IC 50 The value is 22.01+/-0.38 mu M, and the activity is general compared with the positive control of 1.01+/-0.48 mu M. Monomers 8,9 were less active on all four cell lines.
Determination of bacteriostatic Activity of Compounds of example 6
The compound was detected for tetracoccus (Mlicrococcus tetragenus) by a filter paper diffusion method, and the compound and gentamicin sulfate (positive control) were dissolved in acetone at a concentration of 6mg/mL, and sterilized by filtration through a 0.22 μm organic phase microporous filter. Tetracoccus (Mlicrococcus tetragenus) was inoculated into LB solid medium by streaking, and cultured at 37℃for 24 hours for activation. 1mL of sterile water is taken in a 1.5mL centrifuge tube, a small amount of bacteria is dipped in the sterile water by an inoculating loop and is uniformly mixed in the sterile water to prepare bacterial suspension, the concentration of the bacterial suspension is regulated to about 106 cfmu/mL by a contrast turbidity tube, and then a single-hole puncher with the hole diameter of 6mm is used for punching filter paper into a plurality of filter paper discs for sterilization. Adding 100 μl of bacterial suspension into 100ml of LB solid medium at 50-60deg.C, mixing, pouring into a plate, after the medium is solidified, carefully attaching sterile filter paper onto the medium, dropwise adding 5 μl of metabolite or gentamicin sulfate per filter paper sheet, culturing at 37deg.C for 24 hr, measuring diameter of inhibition zone by cross-over method, and repeating three times per group
TABLE 5 diameter of inhibition zone (mm) of Streptomyces sp BYF-112 secondary metabolite 11 against Streptococcus tetratis
As shown in Table 5, the average diameter of the inhibition zone of Mlicrococcus tetragenus by Compound 11 at the test concentration of 30. Mu.g was 14.1mm, which shows a moderate inhibition ability as compared with the positive control gentamicin sulfate of 28.0 mm.
Example 7 Compounds for barnyard grass root growth inhibition assay
The inhibitory activity of the compounds on barnyard grass root growth was determined using a filter paper-petri dish method. The inhibition activity of the crude extract on the root growth of amaranthus retroflexus and common crops is measured by adopting a filter paper-culture dish method. The specific operation is as follows: (1) Soaking barnyard grass in sodium hypochlorite for 15min, and washing with clear water for 2-3 times. The treated seeds were placed in an aqueous filter paper-petri dish, germinated in an illumination incubator (27 ℃ C., 80% relative humidity, 12/12 day/night) for 1d, seeds floating on the water were removed, and the remaining seeds were rinsed clean with distilled water for use. (2) And (3) taking a proper amount of acetone to fully dissolve the crude extract to be tested, preparing a sample solution with the concentration of 100 mug/mL, taking 5mL of the sample solution, uniformly spraying the sample solution into a culture dish paved with filter paper, fully saturating the filter paper and the liquid medicine, and adding 5mL of sterile water after the acetone fully volatilizes. The blank was treated with acetone as the same treatment and 2,4-D at the same concentration as the positive control. (3) The test seeds (20 grains per dish) just barely exposed were selected and placed uniformly in the treated filter paper-petri dishes and incubated in a climatic incubator at 27℃under 80% relative humidity and timed light (12/12 day/night) conditions for 4d. (4) The root length was measured and the herbicidal activity of the compounds was expressed as average root growth inhibition, 3 dishes for each treatment. Average growth inhibition (%) = [ control average root length-treatment average root length ]/control average root length x 100%.
As shown in FIG. 30, monomer compounds 11 and 5 can effectively inhibit barnyard grass root growth at a concentration of 100 μg/mL, the inhibition rates reach 55.3% and 41.7%, respectively, and good inhibition ability is shown; compound 6 has weak inhibition activity on barnyard grass root growth at high concentration, the inhibition rate is 6.1%, and positive control 2,4-Dichlorophenoxyacetic acid can basically inhibit barnyard grass root growth at 100 mug/mL.

Claims (4)

1. Streptomyces aviruleus (Streptomyces tanashiensis) BYF112, which is preserved in China Center for Type Culture Collection (CCTCC) with a preservation number of CCTCC M2019474 in the year 6 and 20 of 2019.
2. The preparation method of the alkaloid compound is characterized by comprising the following steps:
1) Inoculating the streptomyces BYF112 of claim 1 to a Gao's medium, and culturing for 2-3 days at 160-200rpm and 27.5-28.5 ℃ to obtain seed solution;
2) Inoculating the seed liquid into a Gao's culture medium, and fermenting for 6.5-7.5 days at the temperature of 160-200rpm and 27.5-28.5 ℃ to obtain fermentation liquor;
3) Filtering the fermentation liquor obtained in the step 2), extracting the filtrate with ethyl acetate, and concentrating and drying in vacuum to obtain crude extract;
4) Subjecting the crude extract obtained in the step 3) to silica gel column chromatography segmentation, and performing gradient elution by adopting dichloromethane/methanol, wherein the volume ratio of the dichloromethane to the methanol is sequentially 100:0, 100:1, 100:2, 100:4 and 100:8; thereby obtaining 5 elution portions F1 to F5, respectively;
5) Concentrating the component F2 obtained in the step 4), and recrystallizing in methanol to obtain a compound 8; the same treatment is carried out on the obtained component F3 to obtain a compound 11 and a component F3-1, the component F3-1 is concentrated and recrystallized in ethyl acetate to obtain a compound 5 and a compound 10 and a component F3-1-1 respectively, and the component F3-1-1 is separated by gel column chromatography to obtain a compound 2; the obtained component F4 is treated in the same way to obtain a compound 9 and components F4-1, F4-2, F4-3 and F4-4, wherein the components F4-1 and F4-2 are combined and separated by gel column chromatography to obtain compounds 1,6 and 7 respectively, wherein the component F4-3 is recrystallized in methanol after being separated by a silica gel column with methylene dichloride/methanol as an eluent to obtain a compound 3, and the component F4-4 is recrystallized in methanol after being separated by a silica gel column with methylene dichloride/methanol as an eluent to obtain a compound 4; the concentration conditions are as follows: vacuum degree of 0.1 negative pressure, 45 ℃; the compounds 1 and 2 are mauve powders; the compounds 3, 4 and 7 are red powders; the compound 5 is orange yellow powder; the compound 6 is brown powder; the compounds 8 and 10 are colorless crystals; the compound 9 is yellow powder; the compound 11 is dark green powder; compounds 1 to 11 each have the following structural formula:
3. the process for the preparation of compounds 1 to 11 according to claim 2, wherein the fermentation medium comprises the following components/L: KNO (KNO) 3 1g，K 2 HPO 4 ·3H 2 O 0.5g,MgSO 4 ·7H 2 O 0.5g,NaCl 0.5g,FeSO 4 ·7H 2 O0.01 g, soluble starch 20g, H 2 O 1.0L,pH 7.2。
4. Use of a compound 1,2, 5, 6, 11 obtained by the preparation method according to claim 2, characterized in that: wherein the application of the compound 1 in preparing an anti-ovarian cancer drug; the compound 2 can be applied to the preparation of medicaments for resisting human malignant melanoma; the application of the compound 5 in preparing medicines for resisting human malignant melanoma, ovarian cancer and human gastric cancer and inhibiting barnyard grass root growth; application of compound 6 in preparing medicines for resisting human malignant melanoma, ovarian cancer and human gastric cancer and inhibiting barnyard grass root growth; the application of the compound 11 in preparing medicines for resisting malignant melanin, ovarian cancer and gastric cancer of human beings, medicines for inhibiting streptococcus tetrandrus and the application of barnyard grass root growth.
CN202210562230.1A 2022-05-23 2022-05-23 Extraction method and application of alkaloid Active CN115491322B (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
CN202210562230.1A CN115491322B (en) 2022-05-23 2022-05-23 Extraction method and application of alkaloid

Applications Claiming Priority (1)

Application Number Priority Date Filing Date Title
CN202210562230.1A CN115491322B (en) 2022-05-23 2022-05-23 Extraction method and application of alkaloid

Publications (2)

Publication Number Publication Date
CN115491322A CN115491322A (en) 2022-12-20
CN115491322B true CN115491322B (en) 2023-11-03

Family

ID=84465111

Family Applications (1)

Application Number Title Priority Date Filing Date
CN202210562230.1A Active CN115491322B (en) 2022-05-23 2022-05-23 Extraction method and application of alkaloid

Country Status (1)

Country Link
CN (1) CN115491322B (en)

Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2016001410A1 (en) * 2014-07-03 2016-01-07 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Use of metalloproteinase inhibitors against bacterial infections
CN106305793A (en) * 2016-08-29 2017-01-11 佛山市艳晖生物科技有限公司 Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof
CN106497981A (en) * 2016-10-31 2017-03-15 广东省微生物研究所 A kind of method of activating microorganisms recessiveness secondary metabolite biological synthesis gene cluster expression
US10449260B2 (en) * 2011-07-08 2019-10-22 Theodosia Maina-Nock Enhanced in vivo targeting of radiolabelled peptides with the means of enzyme inhibitors

Patent Citations (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US10449260B2 (en) * 2011-07-08 2019-10-22 Theodosia Maina-Nock Enhanced in vivo targeting of radiolabelled peptides with the means of enzyme inhibitors
WO2016001410A1 (en) * 2014-07-03 2016-01-07 Fraunhofer-Gesellschaft zur Förderung der angewandten Forschung e.V. Use of metalloproteinase inhibitors against bacterial infections
CN106305793A (en) * 2016-08-29 2017-01-11 佛山市艳晖生物科技有限公司 Compound bacterial manure used for prevention and control of cotton verticillium wilt and the preparation method thereof
CN106497981A (en) * 2016-10-31 2017-03-15 广东省微生物研究所 A kind of method of activating microorganisms recessiveness secondary metabolite biological synthesis gene cluster expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
Streptomyces xinjiangensis sp. nov., an actinomycete isolated from Lop Nur region;Cong Cheng;Arch Microbiol;第198卷(第8期);全文 *

Also Published As

Publication number Publication date
CN115491322A (en) 2022-12-20

Similar Documents

Publication Publication Date Title
CN107353274B (en) Selenolonic acid I from penicillium oxalicum and application thereof in preparation of human esophageal cancer resistant medicine
CN107298672B (en) Application of seclenic acid I derived from penicillium oxalicum in preparation of anti-human colon cancer drugs
CN111548954B (en) Four anthraquinone compounds and preparation method and application thereof
CN103265550A (en) Alkaloid compound, preparation method thereof, application thereof in preparing paints resisting marine biofouling and anticancer drugs
CN110776518A (en) Azaphilone spiro compounds and preparation method and application thereof
CN115491322B (en) Extraction method and application of alkaloid
CN107298669B (en) Selenolonic acid I from penicillium oxalicum and application of medicine for resisting human oral epidermoid carcinoma
KR19990025976A (en) Use of Seracia marsense strain and prodigiosin isolated from the culture as an immunosuppressive agent
CN110407792B (en) Secalonic acid J compound derived from penicillium oxalicum and preparation method thereof
CN110330544A (en) A kind of bicyclic steroid of 4,4,1- and its preparation method and application
CN114478685B (en) Indole diterpenoid compound and preparation method and application thereof
CN114380782B (en) Compound, preparation method and application of bactericide in preventing and treating rubber anthracnose
CN115287236B (en) Preparation method and application of alkaloid compounds derived from insect symbiotic actinomycetes
CN112795617B (en) Marine fungus secondary metabolite and preparation and application thereof
CN110669800A (en) Application of iso-Penicillium xanthone A from Penicillium oxalicum and adriamycin resistance
CN110407797B (en) Secalonic acid K compound derived from penicillium oxalicum and preparation method thereof
US20100240887A1 (en) New methods of producing HHT
CN107312014B (en) A kind of mould chlorins compound of lattice Féraud and its preparation method and application
CN102898412B (en) Anti-tumor and anti-bacterial dodecacyclo lactone compounds and use thereof
CN104447475B (en) Preparation method and application of penicillenol D1 derived from penicillium citrinum
CN113402471B (en) Siderophore compounds derived from plant endophytic fungi as well as preparation method and application of siderophore compounds
CN115650854B (en) Integrin derivative, its preparation method and application in alpha-glucosidase inhibiting medicine
CN114452309B (en) Poplar leaf and shrubalthea endophytic fungus fermentation extract and preparation method and application thereof
CN115710167B (en) Two diphenyl ether compounds, preparation method and application thereof in preparation of antibacterial drugs
CN104211670A (en) Alkyl pyranone compound and preparation process and application

Legal Events

Date Code Title Description
PB01 Publication
PB01 Publication
SE01 Entry into force of request for substantive examination
SE01 Entry into force of request for substantive examination
GR01 Patent grant
GR01 Patent grant